CN120718146A - Monoclonal antibody against human TNF-beta and use thereof - Google Patents

Abstract

The invention provides a monoclonal antibody of anti-human TNF-beta and application thereof. Antibodies provided include the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOS: 1, 2, and 3 and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOS: 4, 5, and 6, or include the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOS: 7, 8, and 9 and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOS: 10, 11, and 12. The provided antibody can be specifically combined with TNF-beta, can be used for preparing a kit, and has strong specificity and high sensitivity.

Description

Monoclonal antibody against human TNF-beta and use thereof

Technical Field

The invention belongs to the field of biological pharmacy, and in particular relates to an anti-human TNF-beta monoclonal antibody and application thereof.

Background

Tumor necrosis factor-beta (tumor necrosis factor β, TNF- β), also known as lymphotoxin- α (LT- α), is an important member of the TNF superfamily, and is a cytokine produced by lymphocytes, with a broad range of biological functions. Human TNF-beta molecules consist of 205 amino acid residues, signal peptides containing 34 amino acid residues, mature TNF-beta molecules consist of 171 amino acid residues and have a molecular weight of 25kDa.

Human TNF-beta is expressed primarily by lymphocytes, and both antigen and mitogen can stimulate T lymphocytes to secrete TNF-beta. TNF- β mediates a variety of inflammatory, immunostimulatory and antiviral responses, and also relates to the development of secondary lymphoid organs that are cytotoxic to different tumor cells in vitro and in vivo. In addition, TNF- β is involved in the regulation of a variety of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, coagulation and neurotransmission.

In clinical applications, the detection of TNF-beta is of great importance for disease diagnosis and prognosis evaluation. Clinically, the diagnosis, differential diagnosis and prognosis of the relevant diseases can be carried out by measuring the TNF-beta level. Therefore, there is a great need to develop a human TNF- β protein assay kit having excellent performance.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the related art to some extent. The invention provides a monoclonal antibody against human TNF-beta, which can be used as a medicament or used for preparing a detection kit. The anti-human TNF-beta monoclonal antibody provided has excellent performance, can be specifically combined with human TNF-beta, and has high sensitivity, high accuracy and strong specificity. The anti-human TNF-beta monoclonal antibody provided by the application can also be used for developing a diagnosis kit with excellent performance, and is used for diagnosing the content of human TNF-beta in various samples and assisting clinical diagnosis.

Specifically, the invention provides the following technical scheme:

in a first aspect the present invention provides an anti-human TNF- β monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, said antibody or antigen binding fragment thereof being selected from at least one of the following:

(1) Comprises the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO 1, 2 and 3, or a sequence with one or two conservative amino acid mutations with the sequences shown in SEQ ID NO 1, 2 and 3;

And the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOS.4, 5 and 6, or sequences having one or two conservative amino acid mutations with the sequences shown in SEQ ID NOS.4, 5 and 6;

(2) Comprises the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO 7, 8 and 9, or a sequence with one or two conservative amino acid mutations with the sequences shown in SEQ ID NO 7, 8 and 9;

and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOS 10, 11 and 12, or sequences having one or two conservative amino acid mutations with the sequences shown in SEQ ID NOS 10, 11 and 12;

The HCDR 1-HCDR 3 and LCDR 1-LCDR 3 sequences are obtained through a Kabat definition scheme.

In a second aspect the invention provides an anti-human TNF- β monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, said antibody or antigen binding fragment thereof being selected from at least one of the following:

(a) The HCDR1, HCDR and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO. 13 and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO. 14, or

(B) The HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO. 15, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO. 16.

According to an embodiment of the invention, the monoclonal antibody or antigen binding fragment thereof provided above, the sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID No. 13 or 15;

The sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 14 or 16.

According to a specific embodiment of the invention, the sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof provided by the invention is shown in SEQ ID NO. 13, and the sequence of the light chain variable region is shown in SEQ ID NO. 14. According to an embodiment of the invention, the sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof provided by the invention is shown as SEQ ID NO. 15, and the sequence of the light chain variable region is shown as SEQ ID NO. 16.

According to a specific embodiment of the invention, the heavy chain sequence of the provided antibody is shown as SEQ ID NO. 22, and the light chain sequence is shown as SEQ ID NO. 23. According to a specific embodiment of the invention, the heavy chain sequence of the provided antibody is shown as SEQ ID NO. 24, and the light chain sequence is shown as SEQ ID NO. 25.

In a third aspect the invention provides a polynucleotide encoding an anti-human TNF- β monoclonal antibody or antigen binding fragment thereof as described in the first or second aspects above.

According to an embodiment of the invention, the polynucleotide comprises the sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18;

or comprises the sequences shown in SEQ ID NO. 19 and SEQ ID NO. 20.

In a fourth aspect the invention provides a construct comprising a polynucleotide according to the third aspect.

In a fifth aspect the invention provides a host cell comprising a polynucleotide according to the third aspect or a construct according to the fourth aspect.

In a sixth aspect the invention provides a kit comprising an anti-human TNF- β monoclonal antibody or antigen-binding fragment thereof according to the first or second aspect.

The provided kit comprises an anti-human TNF-beta monoclonal antibody or an antigen binding fragment thereof, and can be used for in vitro quantitative analysis of the content of human TGF-beta protein in various biological samples, such as human serum, plasma, cell lysate, cell culture supernatant and urine.

According to an embodiment of the present invention, the kit comprises a first antibody comprising the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOS: 1,2 and 3, and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOS: 4, 5 and 6, and a second antibody comprising the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOS: 7, 8 and 9, and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOS: 10, 11 and 12. The provided kit can simultaneously comprise a first antibody and a second antibody, and can detect a sample by adopting a double-antibody sandwich method ELISA method. One of the antibodies is used as a coating antibody, and the other antibody is used as a detection antibody.

In a seventh aspect, the invention provides a method of detecting anti-human TNF- β content in a biological sample, comprising:

Incubating the anti-human TNF-beta monoclonal antibody or antigen-binding fragment thereof of the first or second aspect with a biological sample, and determining the OD value at a predetermined wavelength after the reaction product;

based on the OD value, TNF- β content in the biological sample is determined.

The biological sample mentioned is an ex vivo sample.

In an eighth aspect, the invention provides a pharmaceutical composition comprising an anti-human TNF- β monoclonal antibody or antigen-binding fragment thereof of the first or second aspect, and a pharmaceutically acceptable carrier.

In a ninth aspect, the invention provides the use of an antibody or antigen binding fragment thereof according to the second aspect of the first aspect above in the manufacture of a kit or medicament for human TNF- β detection.

The beneficial effects obtained by the invention are at least as follows:

The invention provides monoclonal antibodies against human TNF-beta, which antibodies can be used to prepare diagnostic kits. For example, the antibody provided by the invention can be used for combination to detect human TGF-beta protein, and the antibody combination has the characteristics of specificity and high affinity, and the preparation method of the diagnostic detection kit based on the antibody is also provided, so that the specificity of the diagnostic reagent is improved.

Drawings

FIG. 1 shows ELISA detection results of antibodies provided according to an embodiment of the present invention.

Fig. 2 is a standard graph of a kit provided according to an embodiment of the present invention.

Detailed Description

The following detailed description of embodiments of the invention, examples of which are illustrated in the accompanying drawings and, by way of example, are intended to be illustrative, and not to be construed as limiting, of the invention.

As used in this specification and the claims, "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The term "antibody" is used herein in its broadest sense to refer to a protein or polypeptide comprising an antigen binding site or antigen binding fragment or antigen binding portion, and encompasses natural and artificial antibodies of various structures, including but not limited to whole antibody forms or antigen binding fragments of antibodies. According to a specific embodiment, the mentioned antibodies are monoclonal antibodies. The so-called "whole antibody" comprises a protein of at least two heavy chains (H) and two light chains (L) disulfide-linked to each other. Each heavy chain consists of a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH) comprising a heavy chain constant domain CH1, a heavy chain constant domain CH2 and a heavy chain constant domain CH3. Each light chain consists of a light chain variable region (abbreviated VL) and a light chain constant domain CL. The VH and VL regions may be further divided into complementarity determining regions (also known as hypervariable or hypervariable regions, abbreviated CDRs) separated by conserved Framework Regions (FR). Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus (N-terminus) to carboxy-terminus (C-terminus) in the sequence FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively, from the amino terminus, and CDRs of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively, from the amino terminus.

An "antigen binding fragment" is meant to encompass a portion of a full-length antibody that exhibits antigen-binding properties or functions. The term "variable region" or "variable domain" refers to a domain that involves binding of an antigen to an antibody heavy or light chain. As mentioned above, hypervariable or hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen. Examples of antigen binding fragments or antigen binding portions include, but are not limited to, fab ', F (ab ') 2, bispecific Fab ' and Fv fragments (variable regions), linear antibodies, single chain antibodies, single domain antibodies, and the like. The complete antibody is digested with papain to produce two identical antigen binding fragments, called Fab fragments, each of which contains heavy and light chain variable regions and light and heavy chain constant domains CH1.Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain constant domain CH1, including one or more cysteines from the antibody hinge region. The complete antibody was digested with pepsin to obtain F (ab') 2 fragments. The F (ab ') 2 fragment has two antigen binding F (ab) moieties linked together by disulfide bonds, and the F (ab') 2 fragment is a diabody. Single chain antibodies are fusion proteins formed by joining the heavy and light chain variable regions of an antibody via a flexible short peptide of about 10-25 amino acids. Single domain antibodies are antibody fragments consisting of variable domains of a single monomer. Single domain antibodies are also commonly referred to as nanobodies, since they are typically derived from the variable region of the heavy chain of camelid antibodies or shark antibodies. Nanobodies contain only heavy chain CDR regions and are the smallest antigen-binding fragments with complete function. Nanobodies include three CDR regions (CDR 1-CDR 3) and four framework regions FR (FR 1-FR 4). The framework regions FR1, FR2, FR3 and FR4 are separated by complementarity determining regions CDR1, CDR2 and CDR3, respectively. Antigen binding fragments may be obtained recombinantly. For example, a nucleic acid encoding an antigen-binding fragment of interest may be constructed, introduced into an expression vector, and expressed in a suitable host to produce the antigen-binding fragment.

The term "comprising" or "comprises" is intended to include the stated elements or steps but does not exclude other elements or steps. Of course, unless otherwise indicated, inclusion or inclusion also encompasses a situation consisting of the recited elements or steps. For example, when referring to an antibody variable region comprising a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

Reference herein to "affinity" or "binding affinity" is understood in accordance with the ordinary meaning in the art to reflect the strength and/or stability between an antigen and a binding site on an antibody or antigen binding fragment.

"Specific binding" or "specifically binding to", "binding", "specifically targeting" a particular antigen or epitope, or "specific for" a particular antigen or epitope, or "capable of binding" means being distinguished from non-specific interactions, such specific binding can be measured by some method commonly used in the art. The ability of an antibody to bind to an antigen may be measured by an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, the competitive binding of the antibody to be detected and the labeled antibody can be detected by detecting the cells carrying the antigen by flow cytometry and measuring the positive rate index of the cells. The antigen space structure on the cell surface is closer to the form existing in the body, so that the method can reflect the real situation more.

Provided herein, whether an antibody or antigen-binding fragment, or a polynucleotide, is generally isolatable or recombinant. "isolatable" means capable of being identified and isolated and/or recovered from a cell or cell culture expressing a polypeptide or protein. Typically, the isolated polypeptide will be prepared by at least one purification step. By "isolated antibody" is meant that it is substantially free of other antibodies or antigen-binding fragments thereof having different antigen specificities. "recombinant" means that antibodies can be produced in a foreign host cell using genetic recombination techniques.

The CDR sequences of the antibodies shown herein can be obtained in conjunction with existing database analysis. According to a specific embodiment, the CDR sequences of the antibodies provided by the present invention are obtained in combination with the Kabat definition scheme (see, for example, U.S. Dept. Of HEALTH AND Human Servies, "Sequences ofProteins ofImmunological Interest" (1983)). Of course, those skilled in the art can also obtain this by IMGT(Ehrenmann F.,Kaas Q.and Lefranc M.-P.Nucleic Acids Res.,38:D301-D307(2010);Ehrenmann,F.,Lefranc,M.-P.Cold Spring Harbor Protoc.,6:737-749(2011))、Chothia(, for example, see J.Mol. Biol.196:901-917 (1987)) and the like. It will be appreciated by those skilled in the art that differences in the CDRs due to differences in the definition of the methods are also within the scope of the present invention.

Antibodies or antigen binding fragments thereof

The present invention provides an anti-human TNF- β antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, said antibody or antigen binding fragment thereof being selected from at least one of the following:

(1) Comprises the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO 1, 2 and 3, or a sequence with one or two conservative amino acid mutations with the sequences shown in SEQ ID NO 1, 2 and 3;

And the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOS.4, 5 and 6, or sequences having one or two conservative amino acid mutations with the sequences shown in SEQ ID NOS.4, 5 and 6;

(2) Comprises the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO 7, 8 and 9, or a sequence with one or two conservative amino acid mutations with the sequences shown in SEQ ID NO 7, 8 and 9;

And the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOS 10, 11 and 12, or sequences having one or two conservative amino acid mutations with the sequences shown in SEQ ID NOS 10, 11 and 12.

As used herein, a "conservative amino acid mutation" refers to an amino acid mutation that does not cause a significant change in the conformational structure of a protein or polypeptide, while retaining its biological activity. The conservative amino acid mutations mentioned may be mutations between different amino acids having similar physiological and biochemical properties. For example, by conservative amino acid mutations, the provided antibodies still retain specific binding activity for TNF-beta proteins. According to embodiments, the conservative amino acid mutations mentioned may be substitutions, deletions or additions of conservative amino acids. According to specific embodiments, the deletion of a conserved amino acid is one amino acid, two amino acids or three amino acids based on the listed CDR sequences. According to specific embodiments, the conservative amino acid increase is one amino acid, two amino acids or three amino acids increase based on the CDR sequences listed. According to a specific embodiment, "conservative amino acid substitution" means the replacement of another amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative amino acid substitutions may occur between amino acid residues having hydrophobic side chains (e.g., met, ala, val, leu and Ile), between residues having neutral hydrophilic side chains (e.g., cys, ser, thr, asn and gin), between residues having acidic side chains (e.g., asp, glu), between amino acids having basic side chains (e.g., his, lys, and Arg), or between residues having aromatic side chains (e.g., trp, tyr, and Phe). As is known in the art, conservative amino acid substitutions typically do not cause a significant change in the conformational structure of the protein, and thus may preserve the biological activity of the protein. The conservative amino acid substitutions mentioned may be 1 conservative amino acid substitution, 2 conservative amino acid substitutions, 3 conservative amino acid substitutions, etc. The names of amino acids used herein are indicated by single-letter or three-letter codes that are standard in the art. In some embodiments, the provided HCDR sequences have one conservative amino acid substitution with the HCDR sequences shown. In some embodiments, the provided HCDR sequences have a conserved amino acid deletion from the illustrated HCDR sequences. In some embodiments, the HCDR sequences provided have one conservative amino acid increase from the HCDR sequences shown. In some embodiments, the provided LCDR sequences have one conservative amino acid substitution with the illustrated LCDR sequences. In some embodiments, the provided LCDR sequences have one conservative amino acid deletion from the illustrated LCDR sequences. In some embodiments, the provided LCDR sequences have one conservative amino acid increase from the LCDR sequences shown.

The invention also provides a monoclonal antibody or antigen binding fragment thereof, comprising the sequences of HCDR1, HCDR2 and HCDR3 from the heavy chain variable region shown in SEQ ID NO. 13 and the sequences of LCDR1, LCDR2 and LCDR3 from the light chain variable region shown in SEQ ID NO. 14, or comprising the sequences of HCDR1, HCDR2 and HCDR3 from the heavy chain variable region shown in SEQ ID NO. 15 and the sequences of LCDR1, LCDR2 and LCDR3 from the light chain variable region shown in SEQ ID NO. 16. The HCDR sequences and LCDR sequences from these heavy and light chain variable regions may be combined in different defined schemes with minor differences, but are included within the scope of the invention.

According to a specific embodiment, the invention provides an anti-human TNF-beta monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region having a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 13 and a light chain variable region having a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 14. According to a specific embodiment, the invention provides an anti-human TNF-beta monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region having a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 15 and a light chain variable region having a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 16.

As used herein, "sequence identity", "sequence homology", "sequence similarity" refers to the degree to which two polypeptides or proteins or nucleotide sequences are compared and the sequences are identical. To determine the ratio of sequence identity, this can be accomplished in a variety of ways known in the art. Such as may be obtained using published software such as BLAST, ALIGN, BLAST-2. In some embodiments, sequence identity is due to conservative amino acid mutations. According to specific embodiments, the sequence having sequence identity to the heavy chain variable region or the light chain variable region described above may be due to 1 conservative amino acid mutation, 2 conservative amino acid mutations, 3 conservative amino acid mutations, 4 conservative amino acid mutations, 5 conservative amino acid mutations, 6 conservative amino acid mutations, 7 conservative amino acid mutations, 8 conservative amino acid mutations, 9 conservative amino acid mutations, or 10 conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity to the heavy chain variable region or the light chain variable region described above differs in the framework regions.

According to a preferred embodiment, the invention provides an anti-human TNF-beta monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable region having the sequence shown in SEQ ID NO. 13 and a light chain variable region having the sequence shown in SEQ ID NO. 14. According to a preferred embodiment, the invention provides an anti-human TNF-beta monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable region having the sequence shown in SEQ ID NO. 15 and a light chain variable region having the sequence shown in SEQ ID NO. 16.

According to a more preferred embodiment, an anti-human TNF-beta monoclonal antibody or antigen binding fragment thereof is provided comprising a heavy chain as set forth in SEQ ID NO. 22 and a light chain as set forth in SEQ ID NO. 23. According to a more preferred embodiment, an anti-human TNF-beta monoclonal antibody or antigen binding fragment thereof is provided comprising a heavy chain as set forth in SEQ ID NO. 24 and a light chain as set forth in SEQ ID NO. 25.

Polynucleotide

The invention also provides a polynucleotide encoding the anti-human TNF-beta antibody or antigen binding fragment thereof described above. The polynucleotides mentioned are isolatable and include, but are not limited to, DNA, RNA or cDNA, and the like. Isolated polynucleotide sequences may be obtained using methods conventional in the art for encoding the monoclonal antibodies or antibody or antigen binding fragments referred to. According to a specific embodiment, polynucleotides are provided encoding the sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18, which polynucleotide sequences encode the heavy chain variable region shown as SEQ ID NO. 13 and the light chain variable region shown as SEQ ID NO. 14 described above. According to a specific embodiment, polynucleotides are provided encoding the sequences shown as SEQ ID NO. 19 and SEQ ID NO. 20, which polynucleotide sequences encode the heavy chain variable region shown as SEQ ID NO. 15 and the light chain variable region shown as SEQ ID NO. 16 described above.

The term "polynucleotide" as used herein refers to polynucleotides that are not naturally occurring, including polynucleotides isolated from nature (including in vivo) by biological techniques, and also include synthetic polynucleotides. The isolated polynucleotide may be the genome DNA, cDNA, mRNA or other RNA synthesized, or a combination thereof. Provided herein are a plurality of nucleotide sequences encoding the heavy chain variable region and the light chain variable region of an anti-human TNF-beta monoclonal antibody. It is noted that one skilled in the art can design nucleotide sequences that are not exactly identical to the nucleotide sequences provided above, but that all encode the same amino acid sequence, based on codon degeneracy, from the amino acid sequences of the heavy and light chain variable regions provided herein. Such modified nucleotide sequences are also included within the scope of the present invention.

Constructs

To prepare the antibodies of the invention, the polynucleotide sequences encoding the antibodies may be inserted into replicable expression vectors and expressed in host cells or cell-free expression systems. The invention also provides a construct comprising the polynucleotide. A variety of methods commonly used in the art can be used to obtain constructs, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like, for example, polynucleotides can be inserted into expression vectors to form constructs at multiple cloning sites. The construct may contain, as desired, various operators such as promoters, terminators, marker genes, etc., which are operably linked to the polynucleotide. Promoters are typically used to signal the initiation of transcription, and may be selected from lactose promoter (Lac), trp promoter, tac promoter, phage PL and PR promoters as desired, and terminators provide a signal for transcription termination during transcription, and marker genes on constructs are commonly used for screening. Of course, enhancers may also be used as desired to enhance protein expression. The expression vector is not particularly limited, and may be some commercially available expression vectors, or may be an expression vector artificially modified as required, for example, a plasmid, phage, virus, or the like. The virus may be plant cell virus, mammalian cell virus, etc. The construct may express the antibody or protein in vitro, or may be transferred into cells to express the antibody or protein.

Host cells

The invention also provides a host cell containing the polynucleotide or the construct. Any cell suitable for expression of an antibody or protein by a polynucleotide or construct may be used as a host cell. The host cell may be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a yeast cell, a mammalian cell, or the like. Typical host cells may be yeast cells, CHO, HEK-293 cells, COS cells, insect cells of Drosophila S2 or Sf 9. Host cells containing the polynucleotide or construct can be obtained by methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, and the like.

Kit for detecting a substance in a sample

The invention also provides a kit comprising the monoclonal antibody or the antigen binding fragment. The antibody or antigen binding fragment may be present in the kit as a reagent.

Kits are provided that include at least one of the antibodies described above. When only one antibody is present, the biological sample may be directly detected (e.g., by incubating the antibody with the sample and then detecting with a labeled secondary antibody to determine the amount of human TNF- β in the biological sample), and when two antibodies are present, the biological sample may be detected by a double antibody sandwich method (i.e., by sandwich capture and detection of the antigen of interest in the biological sample with two specific antibodies). The double antibody sandwich method is used for detection, and because two specific antibodies are used, target antigens can be effectively captured and detected, false positives and false negatives are avoided, and the double antibody sandwich method is simple to operate and can reduce the influence of human errors although two incubations are needed.

According to particular embodiments, a kit is provided comprising a first antibody comprising the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOS: 1,2, and 3, and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOS: 4, 5, and 6, and a second antibody comprising the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOS: 7, 8, and 9, and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOS: 10,11, and 12. According to a specific embodiment, the first antibody comprises a heavy chain variable region as shown in SEQ ID NO. 13 and a light chain variable region as shown in SEQ ID NO. 14. According to a specific embodiment, the second antibody comprises a heavy chain variable region as shown in SEQ ID NO. 15 and a light chain variable region as shown in SEQ ID NO. 16.

The first antibody may be used as a coating antibody, the second antibody may be used as a detection antibody, or the first antibody may be used as a detection antibody, and the second antibody may be used as a coating antibody.

According to a specific embodiment, the kit comprises a first antibody composition and a second antibody composition,

The first antibody composition comprises a first antibody and a PBS buffer solution of 0.1M-1M (for example, 0.1M-0.5M);

The second antibody composition comprises a biotin-labeled second antibody, 0.1-1M MES buffer (e.g., 0.1-0.5M MES buffer), 0.1-0.5% v/v Tween-20, 0.5-1.0% w/v NaCl, 1-3% w/v BSA, and 0.1-0.5% v/v Proclin-300. According to a preferred embodiment, the second antibody composition comprises a biotin-labeled second antibody, 0.1M MES buffer, 0.2% Tween-20, 0.85% NaCl, 2% BSA and 0.2% Proclin-300.

The kit may also comprise a human TNF-beta standard comprising human TNF-beta having the sequence shown in SEQ ID NO. 21. The purity of the human TNF-beta standard meets certain requirements, such as at least 80%,85%,90%,95%,96%,97%,98%,99%,99.5%, etc. The kit may further include a container, a buffer reagent, etc., as needed. For example, PBS and 1% bovine serum albumin may also be included. These kits can be used in a variety of experimental techniques including, but not limited to, flow cytometry, immunofluorescence, immunohistochemistry, and Western blot. Accordingly, instructions for use may also be included in the kit to facilitate handling and use by those skilled in the art.

To this end, the invention also provides a method for detecting the content of human TNF- β in a biological sample, comprising:

Incubating the anti-human TNF-beta monoclonal antibody or antigen binding fragment thereof and a biological sample, and measuring the OD value at a preset wavelength after the reaction product;

based on the OD value, TNF- β content in the biological sample is determined.

According to a specific embodiment, a method is provided comprising:

1) Coating the pore plate by using a first antibody, and sealing by using a sealing liquid;

2) Adding a human TNF-beta standard or a biological sample, and washing the pore plate after incubation at 37 ℃;

3) Continuing to add the biotin-labeled secondary antibody, and washing the pore plate after incubation at 37 ℃;

4) Continuously adding horseradish peroxidase-labeled streptavidin, incubating at 37 ℃, and flushing the pore plate;

5) Continuously adding color development to develop color, then adding a stopping solution to stop the reaction, detecting reaction products at wavelengths of 450nm and 630nm, and determining an OD value based on the difference value of the reaction products and the stopping solution;

6) And (3) preparing a standard curve based on the OD value of the human TNF-beta standard substance, and determining the content of TNF-beta in the biological sample based on the standard curve.

The provided kit can be used for detecting human TNF-beta by referring to the method.

Herein, reference to biological samples is to ex vivo samples, including but not limited to serum, plasma, cell lysates, cell culture supernatants, urine, and the like.

Method for producing antibodies

The present invention also provides a method for producing the above antibody or antigen binding fragment, comprising culturing the above host cell, and collecting the antibody or antigen binding fragment from the culture.

Antibodies or antigen binding fragments collected from the culture may be purified to obtain a substantially pure product. By "substantially pure" is meant that the antibody or antigen binding fragment is more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, and even more than 99.5%, 99.6%, 99.7%, 99.8% pure.

The technical scheme of the present invention will be described in detail with reference to examples. It should be noted that these examples are only for facilitating understanding of those skilled in the art and should not be construed as limiting the scope of the present invention. The following detailed description of embodiments of the invention is exemplary and intended to be illustrative of the invention and not to be construed as limiting the invention. Unless otherwise indicated, all methods or reagents used in the examples are those commonly used in the art.

Example 1

Example 1 human TNF-beta was determined by the following method and was produced and purified to obtain the corresponding protein.

Firstly, searching human TNF-beta amino acid sequence (shown as SEQ ID NO: 21) on NCBI functional network, adding histidine tag at C end of protein amino acid, optimizing codon in mammal cell expression system by utilizing codon optimizing software, synthesizing expression plasmid, and sequencing to verify gene sequence.

Expression production of human TNF-beta was then performed:

The day before transfection, cells are inoculated into a cell culture bottle at the concentration of 0.8X10 ⁶/mL, cultured in OPM-293CD03 Medium culture solution, placed in a 37 ℃ and 5% CO ₂ incubator until the cell fusion degree reaches 80% -90%, and transfected when the activity is more than 95%. After the TNF-beta expression vector and the transfection reagent PEI are evenly mixed, the mixture is incubated for 15 to 30 minutes at room temperature, the mixture is slowly added into HEK293 cells, and the mixture is continuously cultured in a culture box with 5 percent CO ₂ at 37 ℃. After cell viability <70%, cell cultures were collected, 8000g centrifuged for 10min, supernatants were collected, and cell supernatants were collected by centrifugation at 12000g at 4 ℃ for 30min.

Affinity purification was then performed on the collected cell supernatants:

Cell supernatants were affinity purified by 0.22 μm filter, ni TED-4FF beads pre-loaded gravity column, 20mM PB,300mMNaCl,10% glycerol, 50mM imidazole, pH8.0 solution washed off, 20mMPB,300mM NaCl,10% glycerol, 250mM imidazole, pH8.0 solution eluted, SDS-PAGE running showed >85% purity, protein was dialyzed into 20mMPB,300mM NaCl,10% glycerol, pH8.0 buffer, 0.22 μm filter sterile filtered, BCA assayed protein concentration, 1mg/mL was stored at-20 ℃. The purity of the purified antigen protein is at least more than 80 percent.

Example 2

Example 2 monoclonal antibodies against human TNF-beta were prepared by the following methods, including:

Mixing and emulsifying affinity purified human TNF-beta protein and Freund's adjuvant in equal volume, immunizing rat with 200 mug/dose, raising immunity with 100 mug/dose for 3 weeks, raising immunity for 2 times, each time for 2 weeks, collecting rat blood separating serum, using ELISA to measure serum antibody titer, screening immune rat to make hybridoma fusion experiment, screening and culturing by HAT screening culture medium, using human TNF-beta as ELISA plate to coat antigen, taking hybridoma monoclonal antibody supernatant to make ELISA, screening high-quality positive cell strain, making two rounds of cell subcloning, cloning monoclonal cell strain, named 1A1 and 1F7, and the produced antibodies are 1A1 antibody and 1F7 antibody.

Extracting total RNA of B lymphocyte by TRIzol method, reverse transcribing to cDNA by RT-PCR method, amplifying heavy chain and light chain of antibody by nest PCR, sequencing, respectively connecting the nucleic acid sequences of coding heavy chain and light chain to pCDNA3.4 carrier, transiently transfecting HEK293F cell, purifying cell culture supernatant by protein A column to obtain the antibody for recognizing human TNF-beta.

The heavy chain sequence of the 1A1 antibody is shown as SEQ ID NO. 22, the light chain sequence is shown as SEQ ID NO. 23, the heavy chain variable region sequence is shown as SEQ ID NO. 13, and the light chain variable region sequence is shown as SEQ ID NO. 14 after sequencing. The heavy chain sequence of the 1F7 antibody is shown as SEQ ID NO. 24, the light chain sequence is shown as SEQ ID NO. 25, the heavy chain variable region sequence is shown as SEQ ID NO. 15, and the light chain variable region sequence is shown as SEQ ID NO. 16. The 1A1 antibody showed a purity of >95% on SDS-PAGE and the 1F7 antibody showed a purity of >95% on SDS-PAGE.

Example 3

Example 3 the affinity of the 1A1 antibody and 1F7 antibody prepared in example 2 with antigen was studied. Comprising the following steps:

The human TNF-. Beta.protein prepared in example 1 was used as a coating antigen, incubated at 4℃overnight, and then titered against 2 antibodies, respectively, as follows:

The antigen was diluted to 1. Mu.g/mL, coated onto ELISA plates at 50. Mu.L/well, incubated overnight at 4℃and 1A1 antibody and 1F7 antibody at different dilution factors were added, wherein the control group was incubated with PBS at 37℃for 30min. After washing the plates, horseradish peroxidase (HRP) -labeled goat anti-rat secondary antibody was added at 50. Mu.L/well, incubated at 37℃for 30min, washed, developed and read.

As shown in FIG. 1, the EC50 of the 1A1 antibody was 9.589ng/mL and the EC50 of the 1F7 antibody was 16.24ng/mL.

The results are shown above, which demonstrate that both the 1A1 antibody and the 1F7 antibody show good affinity with the antigen.

EXAMPLE 4 double antibody sandwich ELISA for detecting TNF-beta concentration

Example 4 using the 1A1 antibody and 1F7 antibody obtained in example 2, a kit was prepared based on the principle of the double antibody sandwich method. Comprising the following steps:

(1) 1A1 antibody as coating antibody

The 1A1 antibody prepared in example 2 was transferred to a 14kD dialysis bag, and after sufficient dialysis in PBS buffer, the concentration was adjusted to 2mg/mL for use. The formulation of the antibody preservation solution is 0.1M PBS.

(2) Biotin-labeled 1F7 antibody as detection antibody

Labeling 10mM Biotin (Biotin) and 1F7 antibody at a molecular molar ratio of 20:1 at room temperature for 1-2 hours, transferring the labeling solution to a 14kD dialysis bag after the reaction is finished, performing rotary dialysis in 1 XPBS for 6 times each time for 1-2 hours, taking out the labeling solution after overnight dialysis, testing the protein content in the labeling solution by using a spectrophotometer, calculating and adding a protective solution, fully mixing uniformly, storing at 18-20 ℃, and diluting the protein concentration to 0.5ug/mL by using the labeling preservation solution for later use during testing.

The formulations of the label stock used were 0.1M MES buffer, 0.2% v/v Tween-20, 0.85% w/v NaCl, 2% w/v BSA and 0.2% v/v Proclin-300.

(3) Preparing standard substance

Human TNF-. Beta.antigen was diluted to 0, 15.625, 31.25, 62.5, 125, 250, 500, 1000pg/mL with antigen dilutions, respectively, and dispensed into calibrator tubes.

The diluent formulation used in the dilution of the antigen was 1 XPBS, and 1% w/v BSA.

(4) ELISA detection method evaluation

Coating the coated antibody was diluted to the use concentration (2. Mu.g/mL) with PBS buffer (pH 9.6), 100. Mu.L/well was added to the ELISA plate, and incubated overnight at 4 ℃.

Closing, namely washing the coated micro-pore plate for 4 times in the next day, beating the micro-pore plate on water-absorbing paper, adding 10% new born calf serum, 400 mu L/hole, incubating for 1 hour at 37 ℃, and beating the micro-pore plate on the water-absorbing paper.

Adding standard substance, adding 100 μl protein or serum sample (diluted with 1% bovine serum albumin at a certain concentration). Blank wells were added with 100 μl of 2% neonatal bovine serum. After incubation for 1 hour at 37 ℃, the microwell plates were dried on absorbent paper, washed with TBST wash solution for 4 times, and each wash plate was dried on absorbent paper.

Detection antibody 100. Mu.L of detection antibody diluted with 1% bovine serum albumin was added to each well and incubated at 37℃for 1 hour. The liquid was discarded, and 300. Mu.L TBST wash solution was added to each well to wash the plate 2 times, each time the plate was washed, and the plate was dried on absorbent paper.

Enzyme addition 100. Mu.L of diluted horseradish peroxidase-labeled streptavidin (1:4000 dilution) was added per well and incubated at 37℃for 45 min. The liquid was discarded, and 300 mu LTBST wash solution was added to each well to wash the plate 6 times, each time the plate was washed, and the plate was dried on absorbent paper. To obtain the desired experimental performance, the residual liquid must be thoroughly removed.

Adding substrate for developing, namely adding 50 mu LA solution and 50 mu LB solution into each hole, and incubating for 40 minutes at room temperature and in dark place. Then 50. Mu.L of stop solution was added to each well. The color changes from blue to yellow. If the color is green or the color change is obviously uneven, the plate frame is gently tapped and fully mixed.

Detection reading is that the enzyme label instrument is used for dual-wavelength detection within 30 minutes, and OD value at 450nm of maximum absorption wavelength or 630nm of reference wavelength is measured. The OD value after calibration was measured at 450nm minus the measured at 630 nm. The use of only 450nm measurement results in a higher OD value and reduced accuracy.

FIG. 2 is a graph showing the OD results of detecting different concentrations of TNF-beta by the double-antibody sandwich method in example 4 of the present invention, and as can be seen from FIG. 2, the concentration of the recombinant protein TNF-beta detected by the double-antibody sandwich method of the present invention can be as low as 16pg/mL by the paired anti-TNF-beta monoclonal antibody provided by the present invention, and the detection accuracy is very high.

In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "detailed description," and the like, means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (10)

1. An anti-human TNF- β monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein said antibody or antigen binding fragment thereof is selected from at least one of the following:

(1) Comprises the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO 1, 2 and 3, or a sequence with one or two conservative amino acid mutations with the sequences shown in SEQ ID NO 1, 2 and 3;

And the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOS.4, 5 and 6, or sequences having one or two conservative amino acid mutations with the sequences shown in SEQ ID NOS.4, 5 and 6;

(2) Comprises the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO 7, 8 and 9, or a sequence with one or two conservative amino acid mutations with the sequences shown in SEQ ID NO 7, 8 and 9;

and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOS 10, 11 and 12, or sequences having one or two conservative amino acid mutations with the sequences shown in SEQ ID NOS 10, 11 and 12;

wherein the HCDR 1-HCDR 3 and LCDR 1-LCDR 3 sequences are obtained according to a Kabat definition scheme.

2. An anti-human TNF- β monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein said antibody or antigen binding fragment thereof is selected from at least one of the following:

(a) The HCDR1, HCDR and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO. 13 and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO. 14, or

(B) The HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO. 15, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO. 16.

3. The anti-human TNF-beta monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2,

The heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO. 13 or 15;

the sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID No. 14 or 16;

Optionally, the sequence of the heavy chain variable region is shown as SEQ ID NO. 13, and the sequence of the light chain variable region is shown as SEQ ID NO. 14;

Or the sequence of the heavy chain variable region is shown as SEQ ID NO. 15, and the sequence of the light chain variable region is shown as SEQ ID NO. 16;

Optionally, the antibody or antigen binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO. 22 and a light chain as set forth in SEQ ID NO. 23, or comprises a heavy chain as set forth in SEQ ID NO. 24 and a light chain as set forth in SEQ ID NO. 25.

4. A polynucleotide encoding the anti-human TNF- β monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3;

Optionally, the polynucleotide comprises the sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18;

or comprises the sequences shown in SEQ ID NO. 19 and SEQ ID NO. 20.

5. A construct comprising the polynucleotide of claim 4.

6. A host cell comprising the polynucleotide of claim 4 or the construct of claim 5.

7. A kit comprising the anti-human TNF- β monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3;

optionally, the kit comprises a first antibody comprising the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs 1, 2 and 3, and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs 4, 5 and 6;

the second antibody comprises HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO 7,8 and 9, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NO 10, 11 and 12;

Optionally, the first antibody is a coated antibody and the second antibody is a detection antibody;

optionally, the kit comprises a first antibody composition and a second antibody composition,

The first antibody composition comprises a first antibody and 0.1M PBS buffer;

The second antibody composition comprises a biotin-labeled second antibody, 0.1M MES buffer solution, 0.1% -0.5% v/vTween-20, 0.5% -1.0% w/v NaCl, 1% -3% w/v BSA and 0.1% -0.5% v/v Proclin-300;

Optionally, the kit further comprises a human TNF-beta standard, wherein the human TNF-beta standard comprises human TNF-beta protein, and the sequence of the human TNF-beta protein is shown as SEQ ID NO. 21;

Optionally, the human TNF- β standard has a purity of at least 80%;

optionally, the kit further comprises PBS and 1% bovine serum albumin.

8. A method for detecting human TNF- β content in a biological sample, comprising:

Incubating the anti-human TNF-beta monoclonal antibody or antigen binding fragment thereof according to any one of claims 1-3 with a biological sample, and measuring the OD value at a predetermined wavelength after the reaction product;

Determining the TNF- β content in the biological sample based on the OD value;

Optionally, comprising:

1) Coating the pore plate by using a first antibody, and sealing by using a sealing liquid;

2) Adding a human TNF-beta standard or a biological sample, and washing the pore plate after incubation at 37 ℃;

3) Continuing to add the biotin-labeled secondary antibody, and washing the pore plate after incubation at 37 ℃;

4) Continuously adding horseradish peroxidase-labeled streptavidin, incubating at 37 ℃, and flushing the pore plate;

5) Continuously adding color development to develop color, then adding a stopping solution to stop the reaction, detecting reaction products at wavelengths of 450nm and 630nm, and determining an OD value based on the difference value of the reaction products and the stopping solution;

6) Making a standard curve based on an OD value of the human TNF-beta standard, and determining the content of TNF-beta in the biological sample based on the standard curve;

optionally, the biological sample is selected from at least one of serum, plasma, cell lysate, cell culture supernatant, urine.

9. A pharmaceutical composition comprising the anti-human TNF- β monoclonal antibody or antigen-binding fragment thereof of any of claims 1-3, and a pharmaceutically acceptable carrier.

10. Use of an anti-human TNF-beta monoclonal antibody or antigen binding fragment thereof of any of claims 1-3 in the preparation of a human TNF-beta kit or medicament.